Distal renal tubular acidosis caused by tryptophan-aspartate repeat domain 72 (WDR72) mutations.

Hereditary distal renal tubular acidosis (dRTA) is a rare genetic disease that is caused by mutations in SLC4A1, ATP6V1B1, or ATP6V0A4. However, there are many families with hereditary dRTA in whom the disease-causing genes are unknown. Accordingly, we performed whole exome sequencing and genetic studies of the members of a family with autosomal recessive dRTA of an unknown genetic etiology. Here, we report compound heterozygous pathogenic variations in tryptophan-aspartate repeat domain 72 (WDR72) (c.1777A>G [p.R593G] and c.2522T>A [p.L841Q]) in three affected siblings of a family with dRTA. Both variants segregated with dRTA in the family and were not observed in normal control subjects. Homologous modeling and in silico mutagenesis indicated that R593G and L841Q alter the H-bond formations in the nearby residues, affecting the WDR72 protein structure. All these evidences indicate that the identified WDR72 variations were probably to have caused hereditary dRTA in the reported family. In addition, homozygous nonsense mutation (c.2686C>T [p.R896X]) was identified in another family, strongly supporting the causal role of WDR72 in dRTA. Based on our literature review, WDR72 mutations associated with dRTA have not been previously described. This is the first identification of pathogenic variations in WDR72 as a cause of hereditary dRTA.

Novel ATP6V1B1 and ATP6V0A4 mutations in autosomal recessive distal renal tubular acidosis with new evidence for hearing loss.

Autosomal recessive distal renal tubular acidosis (rdRTA) is characterised by severe hyperchloraemic metabolic acidosis in childhood, hypokalaemia, decreased urinary calcium solubility, and impaired bone physiology and growth. Two types of rdRTA have been differentiated by the presence or absence of sensorineural hearing loss, but appear otherwise clinically similar. Recently, we identified mutations in genes encoding two different subunits of the renal alpha-intercalated cell's apical H(+)-ATPase that cause rdRTA. Defects in the B1 subunit gene ATP6V1B1, and the a4 subunit gene ATP6V0A4, cause rdRTA with deafness and with preserved hearing, respectively. We have investigated 26 new rdRTA kindreds, of which 23 are consanguineous. Linkage analysis of seven novel SNPs and five polymorphic markers in, and tightly linked to, ATP6V1B1 and ATP6V0A4 suggested that four families do not link to either locus, providing strong evidence for additional genetic heterogeneity. In ATP6V1B1, one novel and five previously reported mutations were found in 10 kindreds. In 12 ATP6V0A4 kindreds, seven of 10 mutations were novel. A further nine novel ATP6V0A4 mutations were found in "sporadic" cases. The previously reported association between ATP6V1B1 defects and severe hearing loss in childhood was maintained. However, several patients with ATP6V0A4 mutations have developed hearing loss, usually in young adulthood. We show here that ATP6V0A4 is expressed within the human inner ear. These findings provide further evidence for genetic heterogeneity in rdRTA, extend the spectrum of disease causing mutations in ATP6V1B1 and ATP6V0A4, and show ATP6V0A4 expression within the cochlea for the first time.

A novel splice-acceptor site mutation (IVS13-2A>T) of polycystic kidney disease 1 (PKD1) gene resulting in an RNA processing defect with a 74-nucleotide deletion in exon 14 of the mRNA transcript.

Autosomal dominant polycystic kidney disease (ADPKD) occurs mainly from mutations of polycystic kidney disease 1 (PKD1) gene. A novel mutation of the PKD1 gene due to a nucleotide substitution in splice-acceptor site of IVS13 (AG->TG) was identified by analyses of PKD1-cDNA and genomic DNA. The IVS13-2A>T substitution resulted in an inactivation of this splice site and utilization of cryptic splice acceptor site in exon 14, causing a 74-nucleotide deletion of this exon in the PKD1-mRNA transcript. The abnormal transcript was present ectopically in the patients' lymphocytes. The partial deletion of PKD1-mRNA leads to frameshift translation and introduces a termination signal at codon 1075. The truncated protein with about one quarter of the full-length polycystin-1 is most likely inactive. Thus, the effect of this mutation would be "loss-of-function" type. Allele specific amplification (ASA) was developed to detect the mutation in DNA samples of other family members. The mutation was present in 11 affected but absent in 13 unaffected family members, corresponding to the results of linkage analysis. In addition, it was not observed in DNA samples of 57 unrelated healthy individuals. Hum Mutat 15:115, 2000.

Use of reverse transcription-polymerase chain reaction for cloning of coat protein-encoding genes of cymbidium mosaic virus.

A reverse transcription-polymerase chain reaction (RT-PCR) has been developed for the cloning of coat protein-encoding genes (CP) of cymbidium mosaic virus (CyMV) isolated from three different infected species of Thai orchid: Cattleya, Mokara and Oncidium. The analysis of the compiled sequences of the cDNA clones shows a single open reading frame which encoded a CyMV CP gene. The gene is 669 nucleotides (nt) long and codes for a 23 761-Da protein. The nt sequences of CyMV CP from the Thai isolates showed that some of them differ at a single nt and share 97% homology, but all of them share only 88% homology to the Singapore Oncidium isolate. In addition, the CyMV CP, unlike that from other potexviruses, is distinctive and differs greatly from the amino acid composition deduced from the nt sequence of the CP.

Molecular defect of PKD1 gene resulting in abnormal RNA processing in a Thai family.

Autosomal dominant polycystic kidney disease (ADPKD) is a common human autosomal disorder caused mainly by mutations of the PKD1 gene. In analysis of PKD1 transcripts by long RT-PCR and nested PCR procedures, we observed PKD1-cDNA fragments from three ADPKD siblings from the same family with a size approximately 250 base pairs (bp) shorter than normal. Further investigations showed that the PKD1 transcripts from these patients had been abnormally processed, the nucleotide sequence of exon 43 containing 291 nt was missing from the transcripts, which would result in an abnormal polycystin-1 with an in-frame deletion of 97 amino acids. This splicing defect did not result from a mutation that disrupted the splice donor or acceptor sites adjacent to exon 43 or the branch sites in flanking introns but was most likely due to 20-bp deletion observed in intron 43. The intronic deletion was present in 8 affected members but absent in 11 unaffected members, corresponding with the results of genetic linkage analysis using 5 polymorphic markers in the PKD1 region. Molecular diagnosis of PKD1 in this family could, therefore, be carried out by genomic DNA amplification to directly detect the PKD1 intronic deletion.